Silver nanorods induced oxidative stress and chromosomal aberrations in the Allium cepa model

Abstract

The production of different size and shape silver nanoparticles (AgNPs) has increased considerably in recent years due to several commercial and biological applications. Here, rod‐shaped AgNPs (SNRs) were prepared using the microwave‐assisted method and characterised by ultraviolet–visible spectroscopy, and transmission electron microscopy analysis. The present study aims to investigate the cyto–genotoxic effect of various concentrations (5, 10, and 15 µM) of SNRs using Allium cepa model. As a result, concentration‐dependent cyto–genotoxic effect of SNRs was observed through a decrease in the mitotic index, and an increase in the chromosomal aberrations such as chromosome break, disturbed metaphase, and anaphase bridge. To check the impact of Ag⁺ ions, 15 µM silver nitrate (AgNO₃) was prepared and tested in all the assays. Furthermore, cell viability and different reactive oxygen species assays were performed to test the cytotoxicity evaluation of SNRs. The authors found that in all the tested assays, SNRs at high concentrations (15 µM) and AgNO₃ (15 µM) were observed to cause maximal damage to the roots. Therefore, the current study implies that the cytotoxicity and genotoxicity of SNRs were dependent on the concentration of SNRs.

1 Introduction

There has been a rapid increase in the production and development of nanoparticles (NPs) in various industries due to their diverse properties as compared to the bulk materials [1]. Especially, large volumes of silver NPs (AgNPs) are produced and utilised for many products such as food‐packing films, wound dressing, biomedical devices [2, 3] as well as anti‐inflammatory applications [4]. AgNPs have shape and size‐dependent optical properties, a slight change in the size and shape of the NPs can aid in tuning the optical properties of NPs [5]. Other than spherical AgNPs, rod and triangle shaped‐particles have been utilised for many applications such as sensing and protein interaction studies [6, 7, 8, 9]. Even though the anisotropic AgNPs have better properties than spherical AgNPs, an evaluation of the biocompatibility and toxic effects of anisotropic AgNPs are important.

Various studies have been done to find out the toxic impact of NPs on plant systems [10, 11]. Plants played a significant role in the accumulation and bio‐distribution of many substances released in the environment. Such environmental agents were also very likely to be influenced by AgNPs [12, 13]. Since the 1920s, the Allium cepa (A. cepa) has been one of the simple models used for testing the toxicity of environmental pollutants, and it was approved by the Environmental Protection Agency [14]. The A. cepa test model has often been utilised for the determination of stress‐induced cytotoxicity and genotoxicity for various toxic substances [15, 16]. Advantages to other systems, A. cepa root tip cells have large chromosomes and stains darkly. Hence, these chromosomes and any aberrations therein can be observed clearly through the compound microscope [17, 18]. Furthermore, root cells treated with NPs or toxic metal ions resulted in the obstruction of the normal cell cycle, and this can lead to the formation of different chromosomal aberrations (CAs) [19, 20, 21]. Recently, several reports with regard to the cytotoxic and genotoxic effect of NPs tested on the A. cepa model [14, 22, 23, 24]. The exposure of the A. cepa root tips to copper oxide (CuO) [25] and titanium dioxide (TiO₂) NPs was tested [23]. Recently, Becaro et al. reported that 2 and 8 nm size AgNPs induced a cytotoxic and genotoxic effect on the A. cepa roots [22]. There are other reports with regard to the different concentrations of AgNPs affecting cell division and chromosomal behaviour in the A. cepa model [20, 26, 27]. While the literature is replete with examples of the toxic studies of spherical AgNPs, the effect of other than spherical shaped‐AgNPs has not been explored as much.

In the present work, we aimed to reveal the cytotoxic and genotoxic effect of rod‐shaped AgNPs (SNRs) in the root tips of A. cepa. Initially, microwave‐assisted anisotropic SNRs were synthesised and characterised using ultraviolet (UV)–visible spectroscopy, and transmission electron microscopy (TEM). Then, different concentrations (5, 10 and 15 µM) of SNRs were made to interact with A. cepa roots to analyse the cytotoxic and genotoxic effect of SNRs. The present work revealed a significant and concentration‐dependent reduction in the percentage of the mitotic index (MI), which indicates the cytotoxic potential of SNRs. The genotoxic effects of SNRs were evidenced by an increased percentage of CAs as compared to control. Besides, cell viability assessment, analysis of the generation of reactive oxygen species (ROS), and lipid peroxidation (LPO) assays have been done to check the oxidative stress induced toxicity of SNRs in the A. cepa model.

2 Materials and methods

2.1 Chemicals

Potassium iodide (KI), silver nitrate (AgNO₃), and sodium borohydride (NaBH4) were procured from SRL Pvt. Ltd, India. Hydrogen peroxide (H₂ O₂) was procured from S. D. Fine Chem Limited. Dipotassium hydrogen phosphate (K₂ HPO₄), 2‐deoxy‐D‐ribose, monosodium phosphate, nicotinamide adenine dinucleotide hydrogen (NADH), disodium phosphate, potassium dihydrogen phosphate, tris(hydroxymethyl)aminomethane hydrochloric acid (Tris HCl), trichloroacetic acid (TCA), glacial acetic acid, nitroblue tetrazolium (NBT), sucrose, thiobarbituric acid (TBA), trisodium citrate, and K₂ HPO₄, were procured from Hi‐media Pvt. Ltd, India. Acetocarmine was obtained from Nice chemicals Pvt. Ltd, India.

2.2 Characterisation techniques

The SNRs formation was measured using a UV‐visible spectrophotometer (Evolution 220, Thermo scientific). The aspect ratio and morphology of SNRs were analysed using a high‐resolution transmission electron microscope (FEI Company Tecnai G² 20 Twin). The size and surface charge of SNRs were analysed by using a NanoBrook 90 Plus PALS Particle Size Analyser (Brookhaven Instruments Corporation, USA). The concentration of SNRs was determined by atomic absorption spectroscopy (AAS) (Analyst A 400)

2.3 Synthesis of SNRs

SNRs were prepared by a microwave‐aided method [28]. 27 × 10⁻⁶ M SNRs were synthesised as follows: in a conical flask, a mixture of water (46 ml), 10 mM AgNO₃ (500 µl), 30% H₂ O₂ (120 µl), 60 mM trisodium citrate, and 0.1 M NaBH₄ (200 µl) were added in turn with constant stirring for 5 min. The colour of the reaction mixture was changed to blue. Immediately the blue mixture was placed inside the microwave oven (700 W) for 90 s. The synthesised SNRs were confirmed by the change in the colour of the reaction mixture from blue to red in 90 s. The synthesised SNRs were stored as a suspension at room temperature (RT) (27°C).

2.4 Test system

A. cepa bulbs, weighing 30–35 g each, were purchased from the local market, Vellore. Three A. cepa bulbs were utilised for each concentration of SNRs. Throughout this study along with SNRs, the precursor AgNO₃ (15 µM) was also tested. Each onion bulb was grown in a 50 ml glass beaker filled with distilled water [deionised (DI)] under dark conditions at RT (27°C). The water was changed every 24 h. The whole A. cepa bulb along with roots was directly exposed to test samples (i.e. 5, 10, and 15 µM SNRs, and AgNO₃ (15 µM), respectively, for 4 h at RT. Roots treated with distilled water was used as a control.

2.5 Cell viability assay

The loss of viability of the cells after interaction with SNRs was tested using the Evans blue staining method reported by Baker and Mock [29].

After treatment, root tips were immersed in an Evans blue 0.25% (W/V) dye for 15 min and washed using DI water to remove the excess stain. Then the roots were soaked in 1 ml of N, N ‐dimethylformamide for 1 h at RT (27°C). The extracted colour (blue) was recorded by a UV–visible spectrophotometer (Evolution 220, Thermo scientific) at 600 nm wavelength (λ).

2.6 Optical microscopic evaluation of A. cepa roots

Slides for microscopic analysis were prepared using the acetocarmine squash technique. After 4 h of interaction with the test samples, the roots were washed with DI water, then three root tips were used for each concentration to prepare a slide for microscopic analysis. The treated root tips were hydrolysed in HCl (1 N) for 20 min then stained with acetocarmine stain for 5 min. The alterations in the cell division were examined under a DM‐2500, Leica optical microscope, Germany at 1000× magnification. For each sample, 1000 cells were considered per slide to evaluate the genotoxic effect of SNRs. Three slides were prepared for each treatment condition. The images of cell division phases and CAs were captured by a camera attached to the microscope and processed by Leica‐Application Suite 3.8. Equations (1) and (2) were used to calculate the percentage of mitotic and phase index

$$\mathrm{Mitotic}\mathrm{index}\left(\text{\%}\right)=N_{\mathrm{m}}/N_{\mathrm{t}}\times 100$$ (1)

$$\mathrm{Phase}\mathrm{index}\left(\text{\%}\right)=N_{\mathrm{p}}/N_{\mathrm{t}}\times 100$$ (2)

where N _t is the total number of cells, N _m is the number of total mitotic cells, and N _p is the number of cells in the phase.

2.7 Determination of ROS

2.7.1 Generation of superoxide radical ${(\mathrm{O}}_2^{\cdot })$

The generation of ${\mathrm{O}}_2^{\cdot }$ for the treated and untreated A. cepa roots was determined by Kiba et al. [30]. Fresh roots weighing ≃50 mg were crushed and incubated in the dark for 24 h with 3 ml of Tris–HCl buffer (50 mM, pH 6.5), sucrose (250 mM), NADH (0.2 mM), and NBT (0.2 mM). After 24 h, the amount of ${\mathrm{O}}_2^{\cdot }$ radicals produced by the roots was measured at 530 nm. The generation of ${\mathrm{O}}_2^{\cdot }$ radicals was indirectly measured through NBT which reacts explicitly with ${\mathrm{O}}_2^{\cdot }$ and forms a blue formazan. The concentration of ${\mathrm{O}}_2^{\cdot }$ was calculated using their extinction coefficient of 12.8 mm⁻¹ cm⁻¹ and represented as µmol/g.

2.7.2 Generation of hydrogen peroxide (H₂ O₂)

The level of H₂ O₂ in the treated and untreated roots was measured as reported by Rajeshwari et al. [24]. Fresh 70 mg of the roots were crushed with 5 ml of TCA (0.1%) at 4°C. The crushed samples were centrifuged (at 12,000 rpm for 20 min). H₂ O₂ was indirectly measured based on its potential to oxidise the KI. 0.5 ml potassium phosphate buffer (10 mM, pH 7.0) and 1 ml of KI solution (1 M) were mixed with 0.5 ml of supernatant, and an optical intensity of the reaction mixture was recorded at 390 nm. The H₂ O₂ content in the sample was measured with the help of a standard curve and represented as µmol/g FW.

2.7.3 Estimation hydroxyl radical (•OH)

The •OH produced by the A. cepa samples were measured using a prior method [31]. Fresh roots (1 g) were crushed with 2 ml 2‐deoxyribose which was prepared in 10 mM sodium phosphate buffer (pH 7.4). The crushed samples were centrifuged for 15 min at 12,000 rpm. Then, the supernatant was incubated for 2 h at 37°C. After 2 h, 0.5 ml of supernatant was mixed with 3 ml of TBA (0.5% (w/v)), and 1 ml glacial acetic acid (v/v) and incubated for 30 min at 100°C. After 30 min, the incubated mixtures were allowed to cool down for 10 min before the analysis. The optical density of malondialdehyde (MDA) was recorded at 532 nm and evaluated by its ɛ  = 155 mM⁻¹ cm⁻¹ and represented as µmol/g FW.

2.7.4 Determination of MDA

LPO is a symbol of stress induced cell membrane damage. The fresh SNRs treated and untreated roots (1.5 g) were mixed with 3 ml TBA (0.5%) prepared in TCA (20%) and the whole mixture was crushed using mortar and pestle. The samples were centrifuged for 10 min at 12,000 rpm followed by incubation at 95°C for 30 min. After centrifugation, a clear solution was taken, and its optical density was recorded at 532 and 600 nm. Calculation of the concentration of MDA was done with the help of ɛ  = 155 mM⁻¹ cm⁻¹ [32].

3 Statistical analysis

The obtained mean values (n  = 3) with a standard error of the entire experimental results were statistically analysed using a two‐way analysis of variance. The experimental data were plotted using Graphpad prism.

4 Results and discussion

4.1 Synthesis of SNRs and its characterisation

Microwave‐assisted SNRs were synthesised and their formation was indicated by the dark red colour suspension [6, 28]. The SNRs were further analysed by UV–visible spectroscopy and TEM. The absorption spectra of SNRs have two distinctive surface plasmon resonance (SPR) peaks at the wavelength (λ) of 338 (transverse SPR) and 489 nm (localised SPR) (Fig. 1 a) [6].

Fig. 1.

SNRs prepared by the microwave‐based process

(a) SPR spectra of SNRs, (b) TEM image of SNRs

Furthermore, Fig. 1 b depicts the shape of prepared SNRs characterised through TEM and the average aspect ratio of SNRs was calculated to be 2.7. AAS characterised the concentration of freshly prepared SNRs and the obtained concentration was 27 µM. The mean hydrodynamic diameter (MHD) of as‐synthesised SNRs was 50 ± 0.3 nm. The MHD of different experimental concentrations of SNRs (5, 10, and 15 µM) was found to be similar to as‐synthesised SNRs (Fig. S1 – supplementary information). Agglomeration of SNRs (15 µM) for the experimental period, i.e. 4 h was studied, as result, it did not produce any drastic change on the size of SNRs (Fig. S2 – supplementary information). The obtained net charge of SNRs was −29 ± 0.5 mV. The stabiliser citrate provides stability and a negative charge to the SNRs.

4.2 Microscopic analysis

To the best of our knowledge, this work is the first of its kind wherein the toxic effect of SNRs tested on the root tips of the A. cepa model.

The cytotoxicity and genotoxicity of SNRs were evaluated based on the changes in the chromosomal behaviour and mitotic cell division of A. cepa root tips.

The MI % of A. cepa root tips treated with distilled water (control) was observed to be 16 ± 0.3%. MI % of SNRs treated (5, 10, and 15 µM) root tips decreased significantly (P <0.001) to 11.5 ± 1.1, 6.9 ± 0.4, and 5.4 ± 0.2%, respectively, when compared with the control (Table 1). The MI % was observed to decrease drastically for AgNO₃ (15 µM) as compared to SNRs (15 µM), which was statistically significant to control.

Table 1.

MI (%) of A. cepa root tips cells interacted with SNRs (5, 10, and 15 µM), AgNO₃ (15 µM), and AgNO₃ (released ion concentration from SNRs)

Treatments	Phase index, %				Mean MI %	Mean CA %
	P a	M b	A c	T d
control	76 ± 3	11 ± 1.4	8.4 ± 2.5	4.39 ± 3.2	16 ± 0.3	0
SNRs (5 µM)	65.8 ± 4	13.6 ± 2.3	16.5 ± 4.7	4 ± 0.5	11.54 ± 1.1	3.54 ± 0.1
SNRs (10 µM)	80.8 ± 4.6	6.57 ± 2	10.8 ± 3.6	1.82 ± 1	6.93 ± 0.4	20.56 ± 1.8
SNRs (15 µM)	90.65 ± 2	4.21 ± 1.47	2.19 ± 1.36	0.92 ± 0.5	5.41 ± 0.2	30.69 ± 0.7
AgNO3 (15 µM)	84.49 ± 2.5	4.57 ± 1.12	4.07 ± 1.3	6.84 ± 1.05	4.87 ± 0.24	39.1 ± 4.7

^a Prophase.

^b Metaphase.

^c Anaphase.

^d Telophase.

Generally, MI is used as a cell cycle parameter and a sign of mitotic cell division [33]. Similarly, MI % of A. cepa roots interacted with CuO was reported to decrease with an increase in the concentration of CuO [25], and AgNPs [26, 34, 35]. This decrease in MI % of the root tip cells could be due to the changes in the normal mitotic cell cycle, which blocking the cells from entering prophase [36]. Mohandas and Grant reported that the decrease in the MI % was due to blockage in the G1 phase which tends to suppress DNA synthesis [14]. Cvjetko et al. reported that MI % of AgNO₃ had a significantly higher effect on A. cepa roots as compared to AgNPs [37]. The highest toxicity observed for AgNPs‐CTAB as compared to citrate‐capped AgNPs. In the current work, the abnormalities in the root of A. cepa were dependent on the concentration of SNRs. This implies that the abnormalities that occurred in the roots were dependent on SNR concentration, specifically SNRs at high concentration causes significant changes in the A. cepa roots.

Furthermore, the genotoxic effects of SNRs qualitatively evaluated by studying the CAs in the treated root tips. As per the obtained results, the control (Fig. S3 – supplementary information) did not show any CA (Table 1). Roots treated with SNRs formed different CAs due to the two effects, one is aneugenic, it was characterised by spindle malfunction and second clastogenic breakage in the chromosome during cell division [38].

Figs. 2 and 3 show the different CAs of both clastogenic (chromosome break, anaphase and telophase bridges) and aneugenic (disturbed metaphase, chromosome loss) actions in A. cepa root tips by SNRs (10 and 15 µM).

Fig. 2.

CA induced by SNRs (10 and 15 µM)

(a) Disturbed metaphase, (b) C‐metaphase, (c) Laggard chromosome, (d) Anaphase bridge, (e) Sticky chromosome, (f) Chromosome break or loss

Fig. 3.

CA induced by SNRs

(a) Control anaphase, (b) Anaphase bridge with chromosome loss, (c) Multipolar anaphase, (d) Anaphase bridge, (e) Control telophase, (f) Telophase bridge, (g), (h) Sticky or clumped telophase

The mean CA values of different concentrations of SNRs are shown in Table 1. The average CA % obtained for 15 µM SNRs was much higher than the values found for 5 µM of SNRs

The formation of the bridge was due to the continuous breakage of chromosomes and the reunion of fragmented sections [22]. The improper folding of inner chromosomal chromatin fibre into single chromatids or due to the degradation of chromosomal DNA tends to form sticky chromosomes [39, 40]. It is an irreversible effect and is a common sign of toxic influence on the chromosomes. The disturbed metaphase caused by the depolymerisation of spindle fibres [41]. The chromosomal bridges (anaphase and telophase) in the different phases lead to structural chromosomal mutations [27]. Disruptions in the spindle formation induce the formation of C‐metaphase and C‐mitosis [24]. Furthermore, the microscopic evaluation of AgNO₃ (15 µM) observed to form different chromosome alterations in A. cepa roots (Fig. S4 – supplementary information). The high CA% was obtained for AgNO₃ as compared to SNRs. Cvjetkoa et al. also reported that the toxic effect of AgNO₃ was found to be high as compared to different concentrations of spherical AgNPs on the A. cepa model [37].

4.3 Oxidative stress analysis

4.3.1 Reactive oxygen species analysis (ROS)

The generation of super oxide radicals ${(\mathrm{O}}_2^{\cdot })$ was visually identified by the formation of blue monoformazan [42]. Generation of ${\mathrm{O}}_2^{\cdot }$ for SNRs (5, 10, and 15 µM) treated roots was found to increase significantly 16.2 ± 0.01, 22 ± 0.01, and 26.4 ± 0.02 µmol/g as compared to the control 10.54 ± 0.02 µmol/g (P  < 0.001) (Fig. 4 a). The results obtained from this study implied that SNRs at high concentrations (15 µM) caused more oxidative stress as compared to the low concentration (Fig. 4).

Fig. 4.

Generation ROS

(a) Superoxide radicals (${\mathrm{O}}_2^{\cdot }$), (b) Hydrogen peroxide, (c) Hydroxyl radicals by A. cepa root tips after treated with various concentrations of SNRs (5, 10, and 15 µM), and AgNO₃ (15 µM). Values are means ± SE based on three replicates. *Represents the significance between control and test samples; P  <  0.05

The toxicity of SNRs might be due to the different shapes of NPs as well as the concentration of SNRs; the obtained results supported the microscopic analysis. The hydroxyl radicals (•OH, H₂ O₂) was formed by protonation of superoxide ${(\mathrm{O}}_2^{\cdot })$ in plant cells [43].

Fig. 4 b shows the production of H₂ O₂ in roots (A. cepa) after interaction with the different concentrations of SNRs (5, 10 and 15 µM). The generation of H₂ O₂ was directly proportional to that of the concentration of SNRs. As compared to the control values, the production of H₂ O₂ for the different concentrations of SNRs was non‐significant. However, the production of H₂ O₂ for AgNO₃ (P  < 0.05 was found to be significant to the control values.

Similarly, Fig. 4 c shows that the results of SNRs induced production of •OH in A. cepa roots. As per the results, the generation of •OH for control was found to be statistically significant for higher concentrations of SNRs (10 and 15 µM). The oxidative stress induced by AgNO₃ was found to be less and statistically significant as compared to the control. Thus, the SNRs induced abnormalities in the roots were possibly due to increased generation of mediated by intra‐ or extra‐cellular ROS (${\mathrm{O}}_2^{\cdot }$, H₂ O₂, and •OH) which was confirmed by the microscopic analysis.

It was found that spherical AgNPs, generated different types of ROS which further caused DNA damage and various CAs [10]. The accumulation of NPs in the cytoplasm can lead to a lack of mitochondrial activity, increases in oxidative stress, and cell death [44, 45]. Li et al. reported that the NPs entered into the subcellular organelles such as the nucleus and mitochondria caused an increase in oxidative stress [46]. The mechanism of toxicity of NPs remains unexplored, although it might be due to the major properties such as size, shape, and functional compounds present on the surface of the NPs [5, 47]. The generation of different ROS (${\mathrm{O}}_2^{\cdot }$, •OH, and H₂ O₂) for SNRs was observed to be less as compared to AgNO₃. The production of ROS was observed to be dependent on the concentration of SNRs and it might be responsible for the decreased MI % and increased CA % in the microscopic analysis.

4.3.2 Effect of SNRs on LPO

LPO is one of the major inducers of cytoplasmic membrane damage due to the oxidative stress that occurred in plant cells [16].

The concentrations of malondialdehide (MDA) after exposure to the SNRs (5, 10 and 15 µM) were found to be 1.01 ± 01, 1.15 ± 0.2, and 1.25 ± 0.3 µmol/g. Compared to control (0.67 ± 0.1 µmol/g) the production of MDA for SNRs (5 µM) was non‐significant. Roots treated with AgNO₃ (15 µM) produced significantly (P  < 0.05) increased the level of MDA (Fig. 5).

Fig. 5.

Concentration of MDA generated in roots after interaction with the different concentrations of SNRs (5, 10, and 15 µM) and, AgNO₃ (15 µM) Values are means ± SE based on three replicates. *Represents the significance between control and test samples; P < 0.05

Hence, the results revealed that the SNRs produced fewer LPO as compared to that of AgNO₃. Thus, our LPO results correlate well with the previous results obtained from different ROS assays. Zhornik et al. reported that different concentrations of AgNPs at different concentrations stimulate the process of LPO and it can lead to morphological changes in human lymphocytes [48]. The oxidative stress‐mediated toxicity of NPs was measured indirectly by the interaction between MDA and TBA. After interaction with MDA, the TBA was reduced and resulted in producing a fluorescent adduct [49].

4.3.3 Cell death (Evans blue method)

The cytotoxicity of SNRs treated roots was evaluated to simple Evan's blue staining method. Evans blue dye can penetrate through the membrane of dead cells and stains them. The extracted stain from control roots was found to be less as compared to the SNR (5, 10, and 15 µM). In this study, the SNRs treated root tips showed a concentration‐dependent increase in cell death (Table 2), which was statistically significant (P  < 0.001). As expected, the damage caused by AgNO₃ (15 µM) was found to be high as compared to SNRs.

Table 2.

Cell viability analysis of SNRs in A. cepa roots using Evans blue method

S. No.	Sample	Cell death, %	Cell viability, %
1	control	0	100
2	SNR (5 µM)	7	92.9
3	SNR (10 µM)	19.5	80.5
4	SNR (15 µM)	21.9	78.1
5	AgNO3 (15 µM)	32.03	67.96

5 Conclusion

A finding of the current study established the cytotoxic and genotoxic evaluation of SNRs in the roots tip cells of A. cepa. The experimental data showed a concentration‐dependent generation of different ROS for SNR treated roots, which is indirectly responsible for SNRs induced cyto‐and genotoxicity. The obtained results suggest that the penetration of SNRs to the root cells of A. cepa, induced different ROS generation and could have caused an alteration in the normal mitotic cell division (Fig. 6). The toxic effect of SNRs has been evaluated in terms of a decrease in MI (%) and increased CAs (%). The findings of the present study proved that as an increase in the concentration of SNRs high toxic effect on the roots of A. cepa was established.

Fig. 6.

Schematic representation of SNR‐induced oxidative stress and formation of different CAs